CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.     

In Vitro fertilization failure of normozoospermic men: search for a lack of testicular isozyme of angiotensin-converting enzyme

Background

Angiotensin converting enzyme (ACE) is a metalloprotease with two isoforms. The somatic isoform is a key component of the renin-angiotensin system; its main function is to hydrolyse angiotensin I into angiotensin II. The germinal or testicular isoform (tACE) located at the plasma membrane of the spermatozoa, plays a crucial role in the spermatozoa-oocyte interaction during in vivo fertilization, in rodents. Disruption of the tACE in mice has revealed that homozygous male tACE?/? sire few pups despite mating normally. Few spermatozoa from these tACE?/? mice are bound to the zona pellucida (ZP) despite normal semen parameters. Based on these findings in mice models, we hypothesized that some infertile men that have the same phenotype as the tACE?/? mice, ie normal semen parameters and a lack of sperm bind to the ZP in vitro, may have a tACE defect.

Methods

Twenty four men participated to this study. The case subjects (n?=?10) had normal semen parameters according to the WHO guidelines (WHO 1999) but a total in vitro fertilization failure with absence of sperm fixation to the ZP. The control subjects (n?=?14) also had normal semen parameters and a normal fertilization rate ≥65%. We investigated the tACE expression in spermatozoa by Western-Blot and performed a DNA sequencing of the tACE gene.

Results

Three case-subjects and one control-subject had no tACE expression. There were no statistic differences between the two groups. No mutation was detected in the tACE DNA sequence.

Conclusions

Our results didn’t show any involvement of tACE in human fertilization especially in ZP binding.     

Gene targeting in maize by somatic ectopic recombination

Low transformation efficiency and high background of non‐targeted events are major constraints to gene targeting in plants. We demonstrate here applicability in maize of a system that reduces the constraint from transformation efficiency. The system requires regenerable transformants in which all of the following elements are stably integrated in the genome: (i) donor DNA with the gene of interest adjacent to sequence for repair of a defective selectable marker, (ii) sequence encoding a rare‐cutting endonuclease such as I‐SceI, (iii) a target locus (TL) comprising the defective selectable marker and I‐SceI cleavage site. Typically, this requires additional markers for the integration of the donor and target sequences, which may be assembled through cross‐pollination of separate transformants. Inducible expression of I‐SceI then cleaves the TL and facilitates homologous recombination, which is assayed by selection for the repaired marker. We used bar and gfp markers to identify assembled transformants, a dexamethasone‐inducible I‐SceI::GR protein, and selection for recombination events that restored an intact nptII. Applying this strategy to callus permitted the selection of recombination into the TL at a frequency of 0.085% per extracted immature embryo (29% of recombinants). Our results also indicate that excision of the donor locus (DL) through the use of flanking I‐SceI cleavage sites may be unnecessary, and a source of unwanted repair events at the DL. The system allows production, from each assembled transformant, of many cells that subsequently can be treated to induce gene targeting. This may facilitate gene targeting in plant species for which transformation efficiencies are otherwise limiting.     

Life history of the sandy beach amphipod Deshayesorchestia deshayesii (Crustacea: Talitridae) from Bizerta beach (North of Tunisia)

Population dynamics and reproductive activity of the amphipod Deshayesorchestia deshayesii were studied monthly from June 2007 to December 2008 in Bizerta beach. During this period, 6577 specimens of D. deshayesii were identified; the sex ratio was female biased, with a mean of the monthly sex ratios equal to 0.21?±?0.12. Due to the absence of embryos or eggs in the female brood pouches and juveniles in the samples, results suggested that the reproductive season extended from April to November, with a sexual resting period during winter from December to March. Brood size varied between 4 and 17 eggs and fecundity appeared to be correlated with female size. Six cohorts were identified on the first sampling date. Additionally, 12 new cohorts were detected during the study period. Life span was estimated at 5–7?months depending on the time of birth. Cohorts that appeared at the beginning of the reproductive period tended to have shorter lives than those born later in the season. These results suggest that D. deshayesii can be considered as a semi-annual species, with iteroparous females.     

Cadmium,copper and zinc toxicity effects on growth,proline content and genetic stability of Solanum nigrum L., a crop wild relative for tomato; comparative study

Plants like other organisms are affected by environmental factors. Cadmium, copper and zinc are considered the most important types of pollutants in the environment. In this study, a comparison of growth and biochemical parameters between the crop wild relative (CWR) Solanum nigrum versus its cultivated relative Solanum lycopersicum to different levels of Cu, Zn and Cd stress were investigated. The presence of ZnSO₄ and CuSO₄ in Murashige and Skoog medium affected severely many growth parameters (shoot length, number of roots and leaves, and fresh weight) of both S. nigrum and S. lycopersicum at high levels. On the other hand, CdCl₂ significantly reduced most of the studied growth parameters for both species. S. nigrum exhibited higher tolerance than S. lycopersicum for all types of stress. In addition, results show that as stress level increased in the growing medium, proline content of both S. nigrum and S. lycopersicum increased. A significant difference was observed between the two species in proline accumulation as a result of stress. In addition, a higher accumulation rate was observed in the crop wild relative (S. nigrum) than in cultivated S. lycopersicum. Changes in Inter-simple sequence repeat (ISSR) pattern of CuSO₄ treated S. nigrum and S. lycopersicum plants were also observed. In conclusion, based on growth and biochemical analysis, S. nigrum showed higher level of metals tolerance than S. lycopersicum which indicates the possibility of using it as a crop wild relative for S. lycopersicum.     

Decreasing Tropomyosin Phosphorylation Rescues Tropomyosin-induced Familial Hypertrophic Cardiomyopathy

Studies indicate that tropomyosin (Tm) phosphorylation status varies in different mouse models of cardiac disease. Investigation of basal and acute cardiac function utilizing a mouse model expressing an α-Tm protein that cannot be phosphorylated (S283A) shows a compensated hypertrophic phenotype with significant increases in SERCA2a expression and phosphorylation of phospholamban Ser-16 (Schulz, E. M., Correll, R. N., Sheikh, H. N., Lofrano-Alves, M. S., Engel, P. L., Newman, G., Schultz Jel, J., Molkentin, J. D., Wolska, B. M., Solaro, R. J., and Wieczorek, D. F. (2012) J. Biol. Chem. 287, 44478–44489). With these results, we hypothesized that decreasing α-Tm phosphorylation may be beneficial in the context of a chronic, intrinsic stressor. To test this hypothesis, we utilized the familial hypertrophic cardiomyopathy (FHC) α-Tm E180G model (Prabhakar, R., Boivin, G. P., Grupp, I. L., Hoit, B., Arteaga, G., Solaro, R. J., and Wieczorek, D. F. (2001) J. Mol. Cell. Cardiol. 33, 1815–1828). These FHC hearts are characterized by increased heart:body weight ratios, fibrosis, increased myofilament Ca²⁺ sensitivity, and contractile defects. The FHC mice die by 6–8 months of age. We generated mice expressing both the E180G and S283A mutations and found that the hypertrophic phenotype was rescued in the α-Tm E180G/S283A double mutant transgenic animals; these mice exhibited no signs of cardiac hypertrophy and displayed improved cardiac function. These double mutant transgenic hearts showed increased phosphorylation of phospholamban Ser-16 and Thr-17 compared with the α-Tm E180G mice. This is the first study to demonstrate that decreasing phosphorylation of tropomyosin can rescue a hypertrophic cardiomyopathic phenotype.     

Molecular characterization of extended-spectrum-beta-lactamase-producing Escherichia coli isolates from red foxes in Portugal

The presence of broad-spectrum-cephalosporin-resistant Escherichia coli isolates and the implicated mechanisms of resistance and virulence factor genes were investigated in red fox (Vulpes vulpes) in Portugal. Cefotaxime-resistant E. coli isolates were isolated from two of 52 fecal samples (4 %), being both ESBL producers. The β-lactamase genes found in the two isolates were bla _SHV-12 + bla _TEM-1b. The tet(A) and sul2 genes were also detected in these isolates, together with the non-classical class 1 integron (intI1-dfrA12-orfF-aadA2-cmlA1-aadA1-qacH-IS440-sul3) with the PcH1 promoter. The two isolates belonged to the phylogroup A. Amino acid changes in GyrA (S83L + D87G) and ParC (S80I) proteins were identified in our study. Concerning MLST typing, both isolates were assigned to ST1086, never found before in wild animals, and they presented closely related PFGE patterns. This study reveals the presence of ESBL-producing E. coli isolates, in a wild ecosystem, which could be disseminated through the environment to other niches.